scholarly journals Comparison of the processes involved in reduction by the substrate for two homologous flavocytochromes b2 from different species of yeast

1986 ◽  
Vol 238 (3) ◽  
pp. 745-756 ◽  
Author(s):  
C Capeillère-Blandin ◽  
M J Barber ◽  
R C Bray
Keyword(s):  
Rate Constant ◽  
Lactate Concentration ◽  
Reaction Scheme ◽  
Redox Systems ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rapid Phase ◽  
Redox Species ◽  
Flavocytochrome B2 ◽  
Hansenula Anomala ◽  
Time Courses

A detailed study of the electron exchanges involved between FMN and haem b2 groups within flavocytochrome b2 of yeast Hansenula anomala (H-enzyme) was performed. The results were compared with those for the homologous enzyme of yeast Saccharomyces cerevisiae (Sx-enzyme) re-investigated at 5 degrees C. The mid-point reduction potentials of FMN and haem were determined by two complementary methods: potentiometric titration with substrate, L-lactate, in the presence of dye mediators with quantification of the reduced species performed by spectrophotometry at suitable wavelengths; anaerobic titration of the enzyme by its substrate by monitoring the e.p.r. signals of the semiquinone and Fe3+ species. Values of Em,7 = -19, -23 and -45 V were determined respectively from the data for the three redox systems Ho/Hr, Fo/Fsq and Fsq/Fr in the H-enzyme instead of +6, -44 and -57 mV respectively in the Sx-enzyme [Capeillère-Blandin, Bray, Iwatsubo & Labeyrie (1975) Eur. J. Biochem. 54, 549-566]. Parallel e.p.r rapid-freezing and absorbance stopped-flow studies allowed determination of the time courses of the various redox species during their reduction by L-lactate. The flavin and the haem reduction time courses were biphasic. In the initial fast phase the reduction of flavin monitored by absorbance measurements is accomplished with a rate constant kF = 360 s-1. The reduction of the haem lags the reduction of flavin with a rate constant kH = 170 s-1. The appearance of flavin free radical is slower than the reduction in flavin absorbance and occurs with a rate constant close to that of the reduction of the haem. At saturating L-lactate concentration the initial rapid phase (up to 15 ms) involved in the overall turnover can be adequately simulated with a two-step reaction scheme. The main difference between the enzymes lies especially at the level of the first step of electron exchange between bound lactate and flavin, which for the H-enzyme is no longer the rate-limiting step in the haem reduction and becomes 8-fold faster than in the Sx-enzyme. Consequently in the H-enzyme for the following step, the intramolecular transfer from flavin hydroquinone to oxidized haem, a reliable evaluation of the rate constants becomes possible. Preliminary values are k+2 = 380 s-1 and k-2 = 120 s-1 at 5 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The role of the C-terminal tail of flavocytochrome b2

1989 ◽  
Vol 263 (3) ◽  
pp. 849-853 ◽  
Author(s):  
S A White ◽  
M T Black ◽  
G A Reid ◽  
S K Chapman
Keyword(s):  
Rate Constant ◽  
Structural Integrity ◽  
Lactate Concentration ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Original Activity ◽  
First Order ◽  
Flavocytochrome B2 ◽  
Deactivation Rate ◽  
Deactivation Rate Constant

A flavocytochrome b2 (L-lactate dehydrogenase) mutant was constructed in which the C-terminal tail (23 amino acid residues) had been deleted (Gly-489→Stop). This tail appears to form many intersubunit contacts in the tetrameric wild-type protein, and it was expected that its removal might lead to the formation of monomeric flavocytochrome b2. The isolated tail-deleted mutant enzyme (TD-b2), however, was found to be tetrameric (Mr 220,000). TD-b2 shows Km and kcat. values (at 25 degrees C and pH 7.5) of 0.96 +/- 0.06 mM and 165 +/- 6 s-1 respectively compared with 0.49 +/- 0.04 mM and 200 +/- 10 s-1 for the wild-type enzyme. The kinetic isotope effect with [2-2H]lactate as substrate seen for TD-b2, with ferricyanide as electron acceptor, was essentially the same as that observed for the wild-type enzyme. TD-b2 exhibited loss of activity during turnover in a biphasic process. The rate of the faster of the two phases was dependent on L-lactate concentration and at saturating concentrations showed a first-order deactivation rate constant, kf(deact.), of 0.029 s-1 (at 25 degrees C and pH 7.5). The slower phase, however, was independent of L-lactate concentration and gave a first-order deactivation rate constant, ks(deact.), of 0.01 s-1 (at 25 degrees C and pH 7.5). This slower phase was found to correlate with dissociation of FMN, which is one of the prosthetic groups of the enzyme. Thus fully deactivated TD-b2, which was also tetrameric, was found to be completely devoid of FMN. Much of the original activity of TD-b2 could be recovered by re-incorporation of FMN. Thus the C-terminal tail of flavocytochrome b2 appears to be required for the structural integrity of the enzyme around the flavin active site even though the two are well separated in space.

Polymerization of acrylonitrile initiated by peroxomonosulphate-thiols redox systems

1990 ◽  
Vol 55 (8) ◽  
pp. 2001-2007
Author(s):  
Gurusamy Manivannan ◽  
Pichai Maruthamuthu
Keyword(s):  
Ionic Strength ◽  
Thermodynamic Parameters ◽  
Thermal Polymerization ◽  
Reaction Scheme ◽  
Degree Of Polymerization ◽  
Reducing Agent ◽  
Redox Systems ◽  
Temperature Range ◽  
Rate Of Polymerization

Aqueous thermal polymerization of acrylonitrile (AN) initiated by peroxomonosulphate (HSO5-, PMS)-thiolactic acid (TLA) and PMS-thiomalic acid (TMA) redox systems has been carried out in the temperature range 30-50 °C. The effect of concentration of monomer, initiator, reducing agent, H+, and ionic strength on rate of polymerization, Rp, has been investigated under deaerated conditions. The Rp has been found to depend on, Rp ~ [AN]01.5 [PMS]0.5 [TLA]0.5 in PMS-TLA system and, Rp ~ [AN]02.0 [PMS]1.0 [TMA]0 in PMS-TMA system. The degree of polymerization (Xn) values and thermodynamic parameters have been evaluated. Suitable reaction scheme has been proposed and expressions for Rp and Xn have been obtained.

Maximal Lactate Steady State in Children

1996 ◽  
Vol 8 (4) ◽  
pp. 328-336 ◽  
Author(s):  
Ralph Beneke ◽  
Volker Schwarz ◽  
Renate Leithäuser ◽  
Matthias Hütler ◽  
Serge P. von Duvillard
Keyword(s):  
Steady State ◽  
Lactate Concentration ◽  
Blood Lactate Concentration ◽  
Peak Heart Rate ◽  
Maximal Lactate Steady State ◽  
Specific Test ◽  
Testing Procedures ◽  
Time Courses ◽  
Kinetics Of ◽  
Submaximal Workload

Maximal lactate steady state (MLSS) corresponds to the prolonged constant workload whereby the kinetics of blood lactate concentration clearly increases from steady state. Different results of MLSS in children may reflect specific test protocols or definitions. Three methods corresponding to lactate time courses during 20 min (MLSS I), 16 min (MLSS II), and 8 min (MLSS III) of constant submaximal workload were intraindividually compared in 10 boys. At MLSS I, lactate, V̇O2peak, heart rate, and workload were higher (p < .05) than at MLSS II and at MLSS III. The differences between MLSS I, MLSS II, and MLSS III reflect insufficient contribution to lactate kinetics by testing procedures, strongly depending on the lactate time courses during the initial 10 min of constant workload. Previously published divergent results of MLSS in children seem to reflect a methodological effect more than a metabolic change.

scholarly journals Glutamatergic Neurotransmission and Neuronal Glucose Oxidation are Coupled during Intense Neuronal Activation

2004 ◽  
Vol 24 (9) ◽  
pp. 972-985 ◽  
Author(s):  
Anant B. Patel ◽  
Robin A. de Graaf ◽  
Graeme F. Mason ◽  
Tomoyuki Kanamatsu ◽  
Douglas L. Rothman ◽  
...  
Keyword(s):  
Glucose Oxidation ◽  
Glucose Utilization ◽  
Lactate Concentration ◽  
Sensory Stimulation ◽  
Tca Cycle ◽  
Cortical Activation ◽  
Positron Emission ◽  
Time Courses ◽  
Total Glucose ◽  
The Relationship

13C nuclear magnetic resonance (NMR) experiments have previously shown that glutamatergic neurotransmitter flux (Vcycle(Glu/Gln)) changes proportionately with neuronal glucose oxidation (CMRglc(ox)N) in the nonactivated cortex of anesthetized rats. Positron Emission Tomography measurements of glucose and oxygen uptake during sensory stimulation had shown that the incremental glucose utilization is greater than oxygen leading to the suggestion that the energy required for stimulated neuronal activity arises from nonoxidative glucose metabolism. In this study, the authors used spatially localized 1H-observed, 13C-edited NMR spectroscopy during an infusion of [1,6–13C2]glucose to assess the relationship between changes in Vcycle(Glu/Gln) and glucose utilization (CMRglc(ox)N and CMRglc(nonox)) during the intense cortical activity associated with bicuculline-induced seizures. Metabolic fluxes were determined by model-based analysis of the 13C-enrichment time courses of glutamate-C4 and glutamine-C4 (CMRglc(ox)N, Vcycle(Glu/Gln)) and lactate-C3 (CMRglc(nonox)). The exchange rate between α-ketoglutarate and glutamate was found to be significantly faster than TCA cycle flux both for control (41 μmol·g−1·min−1; 95% CI, 5 to 109 μmol·g−1·min−1) and during seizures (21 μmol·g−1·min−1; 95% CI, 4.4 to 51.8 μmol·g−1·min−1). During seizures, total glucose utilization (CMRglc(ox+nonox)) increased substantially (466% between 0 and 6 minutes; 277% between 6 and 55 minutes). Glucose oxidation (CMRglc(ox)N) also increased (214%; from 0.26 ± 0.02 to 0.57 ± 0.07 μmol·g−1·min−1) but to a lesser degree, resulting in a large increase in cortical lactate concentration. Vcycle(Glu/Gln) increased 233% (from 0.22 ± 0.04 to 0.52 ± 0.07 μmol·g−1·min−1), which was similar to the increase in glucose oxidation. The value of Vcycle(Glu/Gln) and CMRglc(ox)N obtained here lie on the line predicted in a previous study. These results indicate that neuronal glucose oxidation and not total glucose utilization is coupled to the glutamate/glutamine cycle during intense cortical activation.

scholarly journals The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

1974 ◽  
Vol 139 (3) ◽  
pp. 677-697 ◽  
Author(s):  
J. R. Whitaker ◽  
D. W. Yates ◽  
N. G. Bennett ◽  
J. J. Holbrook ◽  
H. Gutfreund
Keyword(s):  
Steady State ◽  
Lactate Dehydrogenase ◽  
Rate Constant ◽  
Active Sites ◽  
Phase 1 ◽  
Lactate Concentration ◽  
Phenol Red ◽  
Phase 2 ◽  
Protein Fluorescence ◽  
Rapid Formation

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6–8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between ENADLactate ⇌ H+ENADHPyruvate. This equilibrium can be altered by changing lactate concentration or pH, and the H+ENADHPyruvate species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s-1. The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The ENADH formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including ENAD+Oxamate and ENADHOxamate' and the abortive ternary complex ENADHLactate. The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells

2012 ◽  
Vol 302 (2) ◽  
pp. C419-C428 ◽  
Author(s):  
Sandrine Genetet ◽  
Pierre Ripoche ◽  
Julien Picot ◽  
Sylvain Bigot ◽  
Jean Delaunay ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Hemolytic Anemia ◽  
Rate Constant ◽  
Rate Constants ◽  
Flow Analysis ◽  
Red Cells ◽  
Stopped Flow ◽  
Loss Of Function ◽  
Dramatic Decrease ◽  
Time Courses

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3transport through the channel and NH3diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rhnulllacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

scholarly journals Analysis of Time Courses of Metabolic Precursors and Products in Heterogeneous Rat Brain Tissue: Limitations of Kinetic Modeling for Predictions of Intracompartmental Concentrations from Total Tissue Activity

1995 ◽  
Vol 15 (3) ◽  
pp. 474-484 ◽  
Author(s):  
Kathleen C. Schmidt ◽  
Günter Mies ◽  
Gerald A. Dienel ◽  
Nancy F. Cruz ◽  
Alison M. Crane ◽  
...  
Keyword(s):  
Rate Constant ◽  
Kinetic Models ◽  
Goodness Of Fit ◽  
Glucose Utilization ◽  
Total Radioactivity ◽  
Tissue Heterogeneity ◽  
Metabolic Products ◽  
Product Loss ◽  
Time Courses ◽  
Total Tissue

The efficacy of various kinetic models to predict time courses of total radioactivity and levels of precursor and metabolic products was evaluated in heterogeneous samples of freeze-blown brain of rats administered [14C]deoxyglucose ([14C]DG). Two kinetic models designed for homogeneous tissues, i.e., a no-product-loss, three-rate-constant (3K) model and a first-order-product-loss, four-rate-constant (4K) model, and a third kinetic model designed for heterogeneous tissues without product loss [Tissue Heterogeneity (TH) Model] were examined. In the 45-min interval following a pulse of [14C]DG, the fit of the TH Model to total tissue radioactivity was not statistically significantly better than that of the 3K Model, yet the TH Model described the time courses of [14C]DG and its metabolites more accurately. The TH- and 4K-Model-predicted time courses of [14C]DG and its metabolites were similar. Whole-brain glucose utilization (CMRglc) calculated with the TH or 3K Model, ∼75 μmol 100 g−1 min−1, was similar to values previously determined by model-independent techniques, whereas CMRglc calculated with the 4K Model was 44% higher. In a separate group of rats administered a programmed infusion to attain a constant arterial concentration of [14C]DG that minimizes effects of tissue heterogeneity as well as any product loss, CMRglc calculated with all three models was 79 μmol 100 g−1 min−1 at 45 min after initiation of the infusion. Statistical comparisons of goodness of fit of total tissue radioactivity were, therefore, not indicative of which models best describe the tissue precursor and product pools or which models provide the most accurate rates of glucose utilization.

Kinetics Of Thrombin, Antithrombin III And Heparin Interactions

1981 ◽  
Author(s):  
S T Olson ◽  
S A Evans ◽  
J D Shore
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Heart Association ◽  
Slow Phase ◽  
Rapid Phase ◽  
Bimolecular Rate Constant ◽  
At Iii ◽  
Active Site Probe ◽  
Initial Binding ◽  
Kinetics Of

The rates of thrombin inactivation by AT III were studied by stopped-flow fluorimetry using p-aminobenzami- dine as an active site probe. p-Aminobenzamidine binds to thrombin with a of KD of 35 μM and a 50-fold fluorescence enhancement. The bimolecular rate constant for the thrombin-AT III reaction was 8.7 × 103 M-1 s-1 at 25° C and pH 7.4, with no diminished amplitude or evidence for a limiting rate at AT III concentrations as high as 10-4 M. Using lower AT III concentrations, an identical bimolecular rate constant was obtained by discontinuous assay of residual thrombin activity. Inactivation of thrombin by antithrombin III complexed with stoichiometric high affinity heparin at concentrations between 2.5 to 20 μM resulted in biphasic reactions: a rapid phase complete in less than 10 milliseconds and a slow phase with a rate constant of 4-6 s-1 . Relative amplitudes and slow phase rate constants showed slight changes over this AT III-heparin concentration range. These results are consistent with weak initial binding of AT III to thrombin in the absence of heparin (KD> 10-4 M), with a major part of the heparin rate enhancement due to a substantial decrease in the KD for the initial binding step. Supported by Michigan Heart Association and USPHS grant F32 HL06171-01.

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